Abstract: Method and system for quantifying target nucleic acids using real-time amplification and internal calibration adjustment. The approach employs a single fixed data point in combination with a single adjustment calibrator amplified on the instrument that is to be calibrated for approximating a complete calibration curve.

Abstract: The present invention provides for a system comprising (a) first polynucleotide encoding a first promoter operatively linked to an open reading frame (ORF) encoding an endoribonuclease, or an enzymatic active fragment thereof, which cleaves a cognition sequence (cog), and (b) a second polynucleotide encoding a second promoter operatively linked to a nucleotide sequence encoding (i) a cog linked to a coding sequence of interest (COI), (ii) a cog linked to the 3? end of a first COI and the 5? end of a second COI, or (iii) a cog locating within a LS (or targeting sequence) linked to a COI.

Abstract: Provided herein, among other things, are various in vitro methods that involve cleaving dsDNA molecules that comprise a mismatched nucleotide using EndoMS. In some embodiments, the method may comprise ligating a T-tailed double-stranded adapter to A-tailed double-stranded fragments of nucleic acid to produce ligation products that comprise adapter-ligated fragments and double-stranded adapter dimers that comprise a T:T mismatch at the ligation junction and cleaving both strands of the adapter dimers using EndoMS.

Abstract: Methods, kits and systems are disclosed for analyzing one or more molecules in a sample. Analyzing the one or more molecules may comprise quantitation of the one or more molecules. Individual molecules may quantitated by PCR, arrays, beads, emulsions, droplets, or sequencing. Quantitation of individual molecules may further comprise stochastic labeling of the one or more molecules with a plurality of oligonucleotide tags to produce one or more stochastically labeled molecules. The methods may further comprise amplifying, sequencing, detecting, and/or quantifying the stochastically labeled molecules. The molecules may be DNA, RNA and/or proteins.

Abstract: The present invention relates to combinations of a linker bridged gene or domain fusion reverse transcriptase enzyme, and more particularly, combinations of a linker bridged gene or domain fusion reverse transcriptase enzyme and their fusion construction utilizing for more efficient and quality DNA synthesis in reverse transcription. The composition of the invention includes a polymerase domain; a linker, consisting of 3-40 amino acids; and an RNase H domain, wherein the RNase H domain is either unmodified or modified with point mutations. The composition may further include another mutated RNase H, a mutated RNase A, and an additional linker which consists of 3-40 amino acids.

Abstract: A mutant MMLV reverse transcriptase that may have an improvement in one or more properties is provided. For example, the present reverse transcriptase is believed to be more efficient relative to other commercially available MMLV reverse transcriptase variants, particularly for templates with a higher GC content.

Abstract: The present invention provides methods for the amplification of nucleic acid molecules. Methods for amplifying target polynucleotides, including mRNA, using oligonucleotides, DNA and RNA polymerases are provided. The invention further provides compositions and kits for practicing the methods, as well as methods which use the amplification products.

Abstract: The present invention relates to the discovery of RNA 5? polyphosphatase enzymes not previously described in the art, methods for discovery of said enzymes, compositions of said enzymes, methods for making said enzymes, and various methods and kits for using said enzymes for biomedical research, for human and non-human diagnostics, for production of therapeutic products, and for other applications. In particular, some embodiments provide compositions, kits and methods for employing RNA polyphosphatases for isolation, purification, production, and assay of capped RNA using a biological sample or a sample from an in vitro capping reaction wherein the sample also contains RNA that is not capped. Other embodiments provide compositions, kits and methods wherein RNA polyphosphatases comprise signal-amplifying enzymes for analyte-specific assays.

Abstract: Provided are methods of adding adapters to nucleic acids. The methods include combining in a reaction mixture a template ribonucleic acid (RNA), a template switch oligonucleotide including a 3? hybridization domain and a sequencing platform adapter construct, a polymerase, and dNTPs. The reaction mixture components are combined under conditions sufficient to produce a product nucleic acid that includes the template RNA and the template switch oligonucleotide each hybridized to adjacent regions of a single product nucleic acid that includes a region polymerized from the dNTPs by the polymerase. Aspects of the invention further include compositions and kits.

Abstract: The present invention relates to kits and methods for efficiently generating 5? capped RNA having a modified cap nucleotide and for use of such modified-nucleotide-capped RNA molecules. In particular, the present invention provides kits and methods for capping RNA using a modified cap nucleotide and a capping enzyme system, such as poxvirus capping enzyme. The present invention finds use for in vitro production of 5?-capped RNA having a modified cap nucleotide and for in vitro or in vivo production of polypeptides by in vitro or in vivo translation of such modified-nucleotide-capped RNA. The invention also provides methods and kits for capturing or isolating uncapped RNA comprising primary RNA transcripts or RNA having a 5?-diphosphate, and methods and kits for using a capping enzyme system and modified cap nucleotides for labeling uncapped RNA comprising primary RNA transcripts or RNA having a 5?-diphosphate with detectable dye or enzyme moieties.

Abstract: The present invention relates to a method for preparing a nucleic acid with high sensitivity, wherein a nucleic acid polymerase is used to add a terminator to the nucleic acid to be used for analysis prior to a nucleic acid polymerization reaction such as a PCR reaction, a real time quantitative PCR reaction, or the like, for detecting a trace of nucleic acid, such that a non-specific priming occurring competitively with an amplification reaction of a target nucleic acid may be basically eliminated, thereby precisely detecting only the trace of target nucleic acid and precisely measuring a concentration of the target nucleic acid.

Abstract: Methods are provided for ligating a 3? adapter and a 5? adapter to a target polynucleotide so as to avoid adapter dimer formation. Embodiments of the methods include adding a blocking oligonucleotide after the first ligation in which a 3? adapter is ligated to the target polynucleotide so that the blocking oligonucleotide is capable of hybridizing to excess 3? adapter and the ligated 3? adapter. Subsequently, a 5? adapter is ligated to the target polynucleotide thus avoiding adapter dimer formation.

Abstract: The present invention is generally directed to compositions, methods, and systems for performing single-molecule, real-time analysis of a variety of different biological reactions. The ability to analyze such reactions provides an opportunity to study those reactions as well as to potentially identify factors and/or approaches for impacting such reactions, e.g., to either enhance or inhibit such reactions. In certain preferred embodiments, RNA templates are used in single-molecule real-time sequencing reactions.

Abstract: The invention relates to a method for linking at least two target nucleic acid molecules from a single biological compartment, comprising the steps of isolating a fraction from a sample, wherein the fraction comprises the compartment comprising at least two nucleic acid molecules; diluting said fraction and aliquoting the dilution in multiple separate reaction vessels such that each reaction vessel comprises preferably one compartment, or encapsulating said compartment in emulsion droplets such that each droplet comprises preferably one compartment; linking said at least two target nucleic acid molecules, preferably by overlap extension PCR. The method may be employed in the analysis of mutations present in a single cell and in the production of antibodies which are present in a single hybridoma.

Abstract: Fusion proteins comprising a single strand DNA binding protein and a nucleic acid polymerase (e.g. DNA polymerase or reverse transcriptase). These high fidelity proteins are suitable for use in nucleic acid amplification methods, including the polymerase chain reaction (PCR).

Abstract: The present invention is in the fields of molecular and cellular biology. The invention is generally related to reverse transcriptase enzymes and methods for the reverse transcription of nucleic acid molecules, especially messenger RNA molecules. Specifically, the invention relates to reverse transcriptase enzymes which have been mutated or modified to increase thermostability, decrease terminal deoxynucleotidyl transferase activity, and/or increase fidelity, and to methods of producing, amplifying or sequencing nucleic acid molecules (particularly cDNA molecules) using these reverse transcriptase enzymes or compositions. The invention also relates to nucleic acid molecules produced by these methods and to the use of such nucleic acid molecules to produce desired polypeptides. The invention also concerns kits comprising such enzymes or compositions.

Abstract: The present invention includes bifunctional shRNAs capable of reducing an expression of a Stathmin 1 gene; wherein at least one target site sequence of the bifunctional RNA molecule is located within the Stathmin 1 gene, wherein the bifunctional RNA molecule is capable of activating a cleavage-dependent and a cleavage-independent RNA-induced silencing complex for reducing the expression level of Stathmin 1.

Abstract: This invention provides a novel method for amplifying and detecting a target gene rapidly with high sensitivity under isothermal conditions. In such method, a sequence to be amplified can be freely designed regardless of the template sequence, an amplified product can be amplified and detected within a short period of time with high sensitivity, and thus, the gene expression level can be determined more easily than is possible with prior art.

Abstract: Methods and compositions relating to the generation and use of gene expression data from tissue samples that have been fixed and embedded are provided. The data can be electronically stored and implemented as well as used to augment diagnosis and treatment of diseases.

Abstract: The present disclosure provides nucleic acids encoding an RNA recognition sequence positioned proximal to an insertion site for the insertion of a sequence of interest; and host cells genetically modified with the nucleic acids. The present disclosure also provides methods of modifying the activity of a target RNA, and kits and compositions for carrying out the methods.

Abstract: The present invention generally relates to methods of making cDNA molecules and cDNA libraries. The invention also relates to cDNA molecules and cDNA libraries produced according to these methods, as well as to vectors and host cells containing such cDNA molecules and libraries. The invention also relates to kits for making the cDNA molecules and libraries of the invention.

Abstract: The present invention relates to kits and methods for efficiently generating 5? capped RNA having a modified cap nucleotide and for use of such modified-nucleotide-capped RNA molecules. In particular, the present invention provides kits and methods for capping RNA using a modified cap nucleotide and a capping enzyme system, such as poxvirus capping enzyme. The present invention finds use for in vitro production of 5?-capped RNA having a modified cap nucleotide and for in vitro or in vivo production of polypeptides by in vitro or in vivo translation of such modified-nucleotide-capped RNA. The invention also provides methods and kits for capturing or isolating uncapped RNA comprising primary RNA transcripts or RNA having a 5?-diphosphate, and methods and kits for using a capping enzyme system and modified cap nucleotides for labeling uncapped RNA comprising primary RNA transcripts or RNA having a 5?-diphosphate with detectable dye or enzyme moieties.

Abstract: The present invention includes assays and kits for detecting the assembly of an RNA binding protein-RNA complex and for detecting the activity of an RNA binding protein.

Abstract: The identification and evaluation of mRNA and protein targets associated with mRNP complexes and implicated in the expression of proteins involved in common physiological pathways is described. Effective targets are useful for treating a disease, condition or disorder associated with the physiological pathway.

Abstract: This invention relates to the use of tumor-derived or associated extracellular ribonucleic acid (RNA) found circulating in blood plasma or serum fraction for the detection, monitoring, or evaluation of cancer or premalignant conditions. Specifically, this invention enables the extraction of circulating RNA from plasma or serum and utilizes nucleic acid amplification assays for the identification, detection, inference, monitoring, or evaluation of any neoplasm, benign, premalignant, or malignant, in humans or other animals, which might be associated with that RNA. Further, this invention allows the qualitative or quantitative detection of tumor-derived or associated extracellular RNA circulating in the plasma or serum of humans or animals with or without any prior knowledge of the presence of cancer or premalignant tissue.

Abstract: Disclosed are methods and compositions for detection and amplification of nucleic acids, wherein two DNA strands hybridized to an RNA strand are ligated. In one aspect, the disclosed methods include removal of an energy source, such as ATP, upon charging a ligase to form an enzyme-AMP intermediate, and then adding substrate, which results in one complete round of RNA-templated DNA ligation. In another aspect, the ligation reaction is accomplished by use of a mixture of at least two different ligase enzymes. The disclosed methods and compositions for RNA-templated DNA ligation may be particularly useful for detection of RNA sequence variants, for example RNA splice variants, and for quantitative expression analysis.

Abstract: The subject of the invention is a peptide with the enzymatic activity of a Dicer-like protein, a method for preparing short RNA molecules, and use thereof. The purpose of the solution was to develop a new method of producing short RNA molecules, using a new, MtDCL1pepA peptide of a Dicer protein activity designed by inventors.

Abstract: A method for designing a bi-shRNA expression cassette encoding a bi-shRNA comprising: selecting one or more target site sequences; providing a backbone sequence comprising a first and a second stem-loop structure, inserting a first passenger strand and a second passenger strand and providing for synthesis of the bi-shRNA expression cassette.

Abstract: Disclosed are DNA polymerases having increased reverse transcriptase efficiency relative to a corresponding, unmodified polymerase. The polymerases are useful in a variety of disclosed primer extension methods. Also disclosed are related compositions, including recombinant nucleic acids, vectors, and host cells, which are useful, e.g., for production of the DNA polymerases.

Abstract: Based at least in part on an understanding of the mechanisms by which small RNAs (e.g., naturally-occurring miRNAs) mediate RNA silencing in plants, rules have been established for determining, for example, the degree of complementarity required between an RNAi-mediating agent and its target, i.e., whether mismatches are tolerated, the number of mismatches tolerated, the effect of the position of the mismatches, etc. Such rules are useful, in particular, in the design of improved RNAi-mediating agents which allow for more exact control of the efficacy of RNA silencing.

Abstract: An HCV/GBV-B chimeric virus which maintains the replication function of HCV and is capable of infecting tamarin is disclosed in order to construct an HCV animal model which can be used as a development or evaluation system for therapeutic agents for HCV. The HCV/GBV-B chimeric RNA comprises an RNA of hepatitis C virus and an RNA of GB virus-B, wherein the RNA of hepatitis C virus comprises an RNA encoding leucine at the 1804th position and lysine at the 1966th position in the amino acid sequence of the polyprotein of hepatitis C virus.

Abstract: The invention features ABC1 nucleic acids and polypeptides for the diagnosis and treatment of abnormal cholesterol regulation. The invention also features methods for identifying compounds for modulating cholesterol levels in an animal (e.g., a human).

Abstract: A nucleic acid extraction device includes a tube that is internally provided with, in the following order, a first plug composed of a first oil, a second plug composed of a first washing liquid, which is phase-separated from an oil and is used for washing a nucleic acid-binding solid-phase carrier having nucleic acids bound thereto, a third plug composed of a second oil, a fourth plug composed of a reverse transcription reaction solution, which is phase-separated from an oil and is used for performing a reverse transcription reaction, a fifth plug composed of a third oil, a sixth plug composed of an eluent, which is phase-separated from an oil and is used for eluting the nucleic acids from the nucleic acid-binding solid-phase carrier having nucleic acids bound thereto, and a seventh plug composed of a fourth oil.

Abstract: Methods for preparing strand-specific sequencing libraries of oligonucleotides using an RNA polymerase promoter to re-transcribe antisense cDNA which has been reverse transcribed from mRNA are provided. The transcription step linearly amplifies sRNA prior to production of double-stranded cDNA to be sequenced and may be sufficient to eliminate the conventional PCR amplification step prior to sequencing. The methods incorporate anchor sequences, amplification sequences and other sequences required for a particular sequencing system or reaction by hybridization and extension of primers, and transcription of RNA, rather than ligation, thus reducing the number of steps and the time required for sample preparation for sequencing of RNA. Use of primer hybridization and transcription reactions in the methods also results in a library that exhibits reduced 3? sequence bias.

Abstract: Disclosed are DNA polymerases having increased reverse transcriptase efficiency relative to a corresponding, unmodified polymerase. The polymerases are useful in a variety of disclosed primer extension methods. Also disclosed are related compositions, including recombinant nucleic acids, vectors, and host cells, which are useful, e.g., for production of the DNA polymerases.

Abstract: The present invention relates to assays and kits for carrying out said assays for the rapid, automated detection of infectious pathogenic agents and normal and abnormal genes. The present invention further relates to methods for general amplification of total mRNAs and for analyzing differential mRNA expression using the amplification methods disclosed herein.

Abstract: The present invention relates to assays and kits for carrying out said assays for the rapid, automated detection of infectious pathogenic agents and normal and abnormal genes. The present invention further relates to methods for general amplification of total mRNAs and for analyzing differential mRNA expression using the amplification methods disclosed herein.

Abstract: A method of processing a target RNA is provided. In certain embodiments, this method comprises: contacting the products of an RNA ligase-mediated ligation reaction with an CAS6 protein, wherein: (i) the RNA ligase-mediated ligation reaction comprises: a target RNA, an RNA ligase, and first and second adaptors that can ligate together to produce an adaptor dimer that contains a CRISPR stem loop; and (ii) the CAS6 protein recognizes the CRISPR stem loop; thereby preventing the adaptor dimer from being reverse transcribed.

Abstract: The present invention relates to a method for detecting and/or for quantifying poly(A) RNA and/or mRNA, wherein a poly(dT) oligonucleotide which features a fluorescent dye and also a quencher is hybridized to the nucleic acid to be detected. Non-hybridized poly(dT) oligonucleotide is degraded by an added nuclease, and as a result, fluorescently labelled nucleotides are released and the resulting fluorescent signal is measured.

Abstract: The nucleotide sequence of a 992 bp region of cDNA and the nucleotide sequence of a 1973 bp (or a 1913 bp) of genomic DNA of the Gr-cm-1 gene were determined for G. rostochiensis. PCR primers and probes specific for G. rostochiensis and G. pallida were generated. PCR assays, including a real-time TaqMan PCR were used to identify G. rostochiensis and G. pallida and to differentiate G. rostochiensis from G. pallida. Transgenic hairy roots expressing Gr-cm-1 dsRNA were generated. There was a 52% reduction in the average number of females per root in the Gr-cm-1 dsRNA transgenic lines when compared with the infected control lines.

Abstract: Compositions of novel polymerase variants and methods of identifying, making and using these novel polymerases are described. The variants have been shown to have advantageous properties such as increased thermostability, deoxyuridine nucleoside triphosphate tolerance, salt tolerance, reaction speed and/or increased reverse transcriptase properties. Uses for these improved enzymes have been demonstrated in isothermal amplification such as LAMP. Enhanced performance resulting from the use of these variants in amplification has been demonstrated both in reaction vessels and in dedicated automated amplification platforms.

Abstract: SARS (severe acute respiratory syndrome virus, a coronavirus) immunogens, antigens, or epitopes, nucleic acid molecules encoding such immunogens, antigens, or epitopes; vectors containing such nucleic acid molecules, e.g., viral vectors such as baculovirus vectors, DNA vectors, such as DNA plasmid vectors, e.g., DNA plasmids that express a nucleic acid molecule in a mammalian cell, uses for such immunogens, antigens or epitopes and vectors, e.g., as an active component immunogenic, immunological or vaccine compositions, or to generate antibodies, such as monoclonal antibodies, and methods for making, and using such immunogens, antigens or epitopes, vectors, antibodies, including in methods for eliciting an immunological or immunogenic or vaccine response, as well as in assays or diagnostic kits or methods, are discussed, as well as a seamless fusion of sequences in a plasmid or vector, e.g., a sequence encoding a leader sequence and a sequence encoding a protein, epitope or immunogen or antigen.

Abstract: Methods for making cDNA molecules, for amplification of RNA by PCR and for preparation of cDNA libraries are provided. Kits for making cDNA molecules also are provided. Compositions are also provided comprising mixtures of reagents, including reverse transcriptases, buffers, cofactors and other components, suitable for immediate use in conversion of RNA into cDNA and RT PCR without dilution or addition of further components. These compositions are useful, alone or in the form of kits, for cDNA synthesis or nucleic acid amplification (e.g., by the Polymerase Chain Reaction) or for any procedure utilizing reverse transcriptases in a variety of research, medical, diagnostic, forensic and agricultural applications.

Abstract: Disclosed are DNA polymerases having increased reverse transcriptase efficiency relative to a corresponding, unmodified polymerase. The polymerases are useful in a variety of disclosed primer extension methods. Also disclosed are related compositions, including recombinant nucleic acids, vectors, and host cells, which are useful, e.g., for production of the DNA polymerases.

Abstract: Disclosed are DNA polymerases having increased reverse transcriptase efficiency relative to a corresponding, unmodified polymerase. The polymerases are useful in a variety of disclosed primer extension methods. Also disclosed are related compositions, including recombinant nucleic acids, vectors, and host cells, which are useful, e.g., for production of the DNA polymerases.

Abstract: The invention relates to methods for the detection of a specific sequence of RNA in a cell or tissue sample. The invention also relates to methods to enzymatically manipulate the RNA in a crude cell lysate in a number of applications.

Abstract: The present invention is a method of generating a library of expressed cDNA sequences using multi-targeted primers that are complementary to degenerate motifs present in a large proportion of corresponding mRNA sequences. Multi-targeting priming (MTP) for genome-wide gene expression assays provides selective targeting of multiple sequences and counter-selection against undesirable sequences. Priming with MTPs in addition to oligo-dT can result in higher sensitivity, a greater number of genes whose expression is well measured, a greater number of genes whose differences in gene expression are detected to be statistically, and a greater power to detect meager differences in expression.

Abstract: Provided herein is method comprising: contacting an initial RNA sample containing a population of different RNA molecules with a divalent cation and a set of DNAzymes that are designed to cleave multiple target RNAs in the initial sample, thereby producing a product RNA sample that comprises: a) uncleaved RNA molecules and b) cleaved RNA fragments that contain a 2?,3?-cyclic-phosphate and a 5? hydroxyl as the result of DNAzyme cleavage.

Abstract: This invention relates to the use of tumor-derived or associated extracellular ribonucleic acid (RNA) found circulating in the plasma or serum fraction of blood for the detection, monitoring, or evaluation of cancer or premalignant conditions. Specifically, this invention enables the extraction of circulating RNA from plasma or serum and utilizes nucleic acid amplification assays for the identification, detection, inference, monitoring, or evaluation of any neoplasm, benign, premalignant, or malignant, in humans or other animals, which might be associated with that RNA. Further, this invention allows the qualitative or quantitative detection of tumor-derived or associated extracellular RNA circulating in the plasma or serum of humans or animals with or without any prior knowledge of the presence of cancer or premalignant tissue.

Abstract: The present invention relates to a method for the diagnosis and/or the follow up of the evolution of cancer, which includes the analysis and quantification of over expressed and amplified genes in the plasma/serum of cancer patients or persons suspected to harbor cancer. This is achieved by analyzing together the amount of DNA and RNA of certain genes in the plasma/serum of cancer patients that are the reflection of a gene amplification and/or a gene over expression in comparison to healthy controls.